Rb1 gene induced protein (rb1cc1) and gene

ABSTRACT

To provide a novel gene and protein involved in multidrug resistance in cancer, to elucidate functions of the gene and protein, to provide methods of detecting the gene and antibody against the protein and of testing and diagnosing cancer using the gene and antibody, we found a novel protein (RB1CC1) or polypeptide and gene thereof present in nucleus of human or animal cells and having transcription factor functions and/or functions inducing expression of retinoblastoma-1 gene (RB1 gene) or the gene product. We determined the amino acid sequence and cDNA sequence, conducted gene amplification and detection with primers hybridizing with the gene, tested for expression and mutation of the gene, discovered the gene relates to cancer cell proliferation and assayed cancers, prepared antibody against the protein and detected the protein using the antibody, whereby we found a relation between the protein and cancer cell proliferation, and assayed cancers.

TECHNICAL FIELD

The present invention relates to a novel protein and polypeptide(hereunder, referred to as “novel protein RB1CC1”) that can induceexpression of a tumor-suppressor gene (retinoblastoma gene: RB1 gene).More specifically, the present invention relates to a polypeptide havingall or a part of an amino acid sequence of a novel protein, a nucleicacid (hereunder, referred to as “RB1CC1 gene”) coding for thepolypeptide, a recombinant vector containing the nucleic acid, atransformant that was transformed with the recombinant vector, a methodfor producing a peptide or polypeptide using the transformant, anantibody against the peptide or polypeptide, a method of screening forcompounds that utilizes these, the screened compounds, anactivity-inhibiting compound or activity-enhancing compound that acts onthe polypeptide or the nucleic acid, a pharmaceutical compositionrelating to these, and a method of testing or diagnosing a diseaserelating to these as well as a reagent.

BACKGROUND OF THE INVENTION

Multidrug resistance (MDR) that is resistance to treatment withanticancer agents is a major barrier to the successful treatments ofcancer. While current understanding of factors that contribute toorigins of MDR is limited, it is considered that P-glycoprotein that isa product of an MDR-associated gene (MDR1 gene) is involved in severalcancers. It is also known that in other cancers expression ofP-glycoprotein correlates inversely with emergence and metastasis of thecancer. It is considered that these different effects of P-glycoproteinare subject to suppression by different gene products or conductdifferent interactions. The identification of genes associated with MDRis essential in order to clarify these phenomena.

SUMMARY OF THE INVENTION

A problem to be solved by the present invention is to discover a geneassociated with multidrug resistance to anticancer agents as describedabove and the gene product thereof. More specifically, an object of thepresent invention is to provide a novel protein and polypeptide (novelprotein RB1CC1) that can induce expression of the tumor-suppressor gene(retinoblastoma gene: RB1 gene). Another object of the present inventionis to provide the nucleic acid (hereunder, “RB1CC1 gene”) coding for allor the part of the amino acid sequence of the novel protein, and themethod for producing the protein or polypeptide (novel protein RB1CC1)using genetic engineering techniques. A further object of the presentinvention is to provide the antibody against the polypeptide derivedfrom the novel protein RB1CC1. Other objects of the present inventionare to conduct screening for an inhibitor, antagonist, or activator foractions of the novel protein RB1CC1 utilizing the aforementionedsubstances, to provide screened compounds, and to provide thepharmaceutical composition for use in treatment of multidrug resistance(MDR) that is resistance to treatment with anticancer agents utilizingthese. Another problem to be solved by the present invention is toprovide the method for diagnosing a cancer cells or cancer by testingfor the novel protein and polypeptide (RB1CC1 protein) that can induceexpression of the tumor-suppressor gene (retinoblastoma gene: RB1 gene)or the nucleic acid (hereunder, “RB1CC1 gene”) coding for all or a partof the amino acid sequence of the protein, that were clarified in thepresent invention. A further object of the present invention is toprovide nucleic acid primers that can amplify a nucleic acid coding forall or the part of the amino acid sequence of the protein, and toprovide the method for diagnosing cancer cells or cancer by testing foran amplification product of the nucleic acid using primers. A stillfurther object of the present invention is to provide the antibody thatcan react with the protein or polypeptide (RB1CC1 protein), as well asan immunological assay method that uses the antibody. A further objectof the present invention is to provide an assay reagent or kit that usesthe primers or the antibody to be used in the assay method.

In order to solve the above problems, the present inventors identified agene expressing differentially in U-2 OS osteosarcoma cells andMDR-variant induced cells and determined the nucleotide sequence thereofand the amino acid sequence encoded by cDNA of the novel protein.Further, in order to verify that a similar protein is present inanimals, inventors determined the amino acid sequence of a novel proteinin mouse and the amino acid sequence encoded by cDNA of the novelprotein. In addition, inventors prepared antibodies that recognize theseproteins and conducted immunological assay in addition to assay ofexpression, mutation, deletion and the like for the gene, and found thatexpression of the gene and expression of the protein are suppressed incertain kinds of cancer cells, thereby completing the present invention.

That is, the present invention comprises the following:

-   1. A protein or polypeptide which is present in the nucleus of human    or animal cell and which has a function that can induce a    transcription factor function and/or expression of retinoblastoma    gene (RB1 gene) or a gene product thereof.-   2. The human protein according to the above 1, which is a    polypeptide or protein selected from the group consisting of: (1) a    polypeptide or protein represented by an amino acid sequence    described in SEQ ID No: 1 in the sequence listing; (2) a polypeptide    containing an amino acid sequence comprising at least five amino    acids of the amino acid sequence of the said polypeptide or    protein; (3) a polypeptide or protein having homology of at least    approximately 70% at the amino acid sequence level with the said    polypeptide or protein; and (4) a protein or polypeptide having a    mutation or induced mutation such as a deletion, substitution or    addition of one to several amino acids relative to the amino acid    sequence of the polypeptide or protein according to any one of the    preceding (1) to (3).-   3. The animal protein according to the above 1 that is a protein    derived from mouse, which is a polypeptide or protein selected from    the group consisting of: (1) a polypeptide or protein represented by    an amino acid sequence described in SEQ ID No: 2 in the sequence    listing; (2) a polypeptide comprising at least five amino acids of    the amino acid sequence of the said polypeptide or protein; (3) a    polypeptide or protein having homology of at least approximately 70%    at the amino acid sequence level with the said polypeptide or    protein; and (4) a protein or polypeptide having a mutation or    induced mutation such as a deletion, substitution or addition of one    to several amino acids relative to the amino acid sequence of the    said polypeptide or protein according to any one of the    preceding (1) to (3).-   4. A nucleic acid coding for the polypeptide or protein according to    any one of the above 1 to 3, or a complementary strand thereof.-   5. A nucleic acid hybridizing under stringent conditions with the    nucleic acid or the complementary strand thereof according to the    above 3.-   6. A nucleic acid represented by a base sequence comprising at least    15 consecutive bases of the base sequence of a nucleic acid    described in SEQ ID No: 3 to 4 in the sequence listing or a    complementary strand thereof, wherein a polypeptide expressed by    transcription of the nucleic acid is the polypeptide according to    any one of the above 1 to 3.-   7. A recombinant vector containing the nucleic acid according to any    one of the above 4 to 6.-   8. A transformant that was transformed with the recombinant vector    according to the above 7.-   9. A method for producing the polypeptide or protein according to    any of the above 1 to 3, comprising a step of culturing the    transformant according to the above 8.-   10. Nucleic acid primers represented by SEQ ID Nos: 5 to 132 in the    sequence listing, which hybridize under stringent conditions with    the nucleic acid or the complementary strand thereof according to    any one of the above 4 to 6.-   11. An antibody that immunologically recognizes the polypeptide or    protein according to any one of the above 1 to 3.-   12. A method of screening for compounds that inhibit or enhance a    function that can induce transcription factor activity of the    polypeptide or protein and/or expression of RB1 gene according to    any of the above 1 to 3, wherein the method uses at least one member    of the group consisting of the polypeptide or protein according to    any one of the above 1 to 3 and the antibody according to the above    11.-   13. A method of screening for compounds that interact with the    nucleic acid according to the above 4 or 6 to inhibit or enhance    expression of the nucleic acid, wherein the method uses at least one    member of the group consisting of the nucleic acid according to    anyone the above4 to 6, the vector according to the above 7, the    transformant according to the above 8, and the nucleic acid primers    according to the above 10.-   14. A compound that was screened by the screening method according    to the above 12 or 13.-   15. A compound that inhibits or enhances a function that can induce    transcription factor activity and/or expression of RB1 gene of the    polypeptide or protein according to any of the above 1 to 3.-   16. A compound that interacts with the nucleic acid according to any    one of the above 4 to 6 to inhibit or enhance expression of the    nucleic acid.-   17. A pharmaceutical composition for use in treatment of multidrug    resistance that is resistance to treatment with anticancer agents,    wherein the pharmaceutical composition comprises at least one member    of the group consisting of the polypeptide or protein according to    any of the above 1 to 3, the nucleic acid according to any one of    the above 4 to 6, the vector according to the above 7, the    transformant according to the above 8, the nucleic acid primers    according to the above 10, the antibody according to the above 11,    and the compound according to any one of the above 14 to 16.-   18. A method of testing or diagnosing a disease related with    expression or activity of the polypeptide or protein according to    any of the above 1 to 3, wherein the method comprises a step of    conducting analysis employing (a) a nucleic acid encoding the    polypeptide or protein and/or (b) the polypeptide or protein in a    sample, as a marker.-   19. The method of testing or diagnosing according to the above 18,    which is a method of testing cancer cells or a method of diagnosing    a cancer.-   20. The method according to the above 18 or 19 which examines    expression, increase, decrease, deletion or the like of all or a    part of the polypeptide or protein according to any of the above 1    to 3, wherein the method uses the antibody according to the above    11.-   21. The method according to the above 18 or 19 which examines    expression, mutation, deletion or insertion or the like of all or a    part of a gene encoding the polypeptide or protein according to any    of the above 1 to 3 through a step of amplifying a gene encoding the    polypeptide or protein according to any of the above 1 to 3 using at    least one of nucleic acid primers according to the above 10.-   22. The method according to any of the above 18 to 21, wherein the    method combines examination of expression, increase, decrease,    mutation, deletion or insertion or the like of all or a part of the    tumor-suppressor gene retinoblastoma gene (RB1 gene) or the gene    product thereof (RB1 protein).-   23. The method according to any of the above 18 to 22, wherein the    method combines examination of expression, increase, decrease,    mutation, deletion or insertion or the like of all or a part of    multidrug resistance gene (MDR1 gene) or the gene product thereof    (MDR1 protein: P-glycoprotein).-   24. The method according to any of the above 18 to 23, wherein the    method combines examination of expression, increase, or decrease or    the like of all or a part of the cell proliferation marker, Ki-67    protein.-   25. A method that examines drug sensitivity of a cancer cell using    the method according to the above 23.-   26. A reagent and a kit for assay or diagnosis, for use in the    method according to any of the above 18 to 25.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows photographs of Northern blots that examined the relationbetween expression of human RB1CC1 gene and MDR1 gene.

FIG. 2 shows a photograph of Western blots and of cellularimmunostaining showing that human RB1CC1 protein is present in nucleus.

FIG. 3 shows photographs of Western blots and of cellular immunostainingshowing that mouse Rb1cc1 protein is present in nucleus.

FIG. 4 is a diagram that examined the effect on cell proliferationresulting from treatment with the anticancer agent doxorubicin.

FIG. 5 shows photographs of Northern blots that examined the relationbetween cell proliferation caused by treatment with the anticancer agentdoxorubicin and RB1CC1 gene expression and RB1 gene expression.

FIG. 6 is a photograph of electrophoresis of RT-PCR products thatexamined the relation between RB1CC1 gene expression and RB1 geneexpression in various cancer cells.

FIG. 7 shows photographs of Northern blots that examined the relationbetween RB1CC1 gene expression and RB1 gene expression in various humanorgans.

FIG. 8 is a photograph of a Northern blot that examined the relationbetween RB1CC1 gene expression and RB1 gene expression in various mouseorgans.

FIG. 9 is a photograph of electrophoresis of RT-PCR products thatexamined the effect on RB1 gene expression caused by introduction ofRB1CC1 gene.

FIG. 10 is a diagram showing results obtained after testing the effecton transcriptional activity of RB1 gene promoter region caused by RB1CC1gene induction.

FIG. 11 is a photograph of results obtained after testing loss ofheterozygosity of RB1CC1 gene locus in a variety of primary breastcancers.

FIG. 12 shows a photograph of electrophoresis of RT-PCR products thatexamined mutation of RB1CC1 gene in primary breast cancers, and a viewshowing the results of gene sequence analysis.

FIG. 13 shows photographs of Western blots that examined expression ofRB1CC1 protein and RB1 protein in primary breast cancers.

FIG. 14 shows photographs of immunohistological staining that examinedexpression of RB1CC1 protein and RB1 protein in primary breast cancers.

FIG. 15 shows diagrams illustrating the correlation between RB1CC1 as astain indicator and Ki-67 and RB1.

DETAILED DESCRIPTION OF THE INVENTION

(Novel Protein RB1CC1)

The cDNA of the nucleic acid encoding the novel protein RB1CC1 providedaccording to the present invention was obtained by identifying a geneexpressing differentially in U-2 OS osteosarcoma cells and MDR-variantinduced cells, conducting amplification employing U-2 OS mRNA as atemplate using nucleic acid primers described in SEQ ID Nos: 5 to 37 inthe sequence listing, and determining the amino acid sequence coded forby cDNA of the novel protein and the base sequence, to thereby obtainthe cDNA as a substance having a novel amino acid sequence. The cDNA ofnovel protein RB1CC1 of the present invention had a length of 6.6 kb,included an open reading frame (ORF) of 4782 nucleotides, and encoded aprotein comprising 1594 amino acids with a molecular weight of 180 kDa.

The novel human protein RB1CC1 had a consensus nuclear localizationsignal sequence site (lysine-proline-arginine-lysine sequence: KPRK), aleucine zipper motif sequence site, and a coiled-coil structure. It wassuggested that the novel human protein RB1CC1 has DNA-binding andtranscription functions.

(Novel Mouse Protein Rb1cc1)

Amplification was conducted employing mRNA of mouse muscle as a templateusing the nucleic acid primers described in SEQ ID Nos: 53 to 83 in thesequence listing, and the amplification product was analyzed. Theobtained cDNA coding for novel mouse protein Rb1cc1 had a chain lengthof 6518 bp with an open reading frame (ORF) of 4764 bp encoding 1588amino acids. The novel mouse protein Rb1cc1 gene shared 89% homologywith the novel human protein RB1CC1 gene. Similarly to the humanprotein, novel mouse protein Rb1cc1 had a consensus nuclear localizationsignal sequence site (lysine-proline-arginine-lysine sequence: KPRK), aleucine zipper motif sequence site, and a coiled-coil structure. It wassuggested that mouse novel protein Rb1cc1 also has DNA-binding andtranscription functions.

(Function of Novel Protein and Gene)

To investigate the role of RB1CC1 gene of the present invention in MDR,RB1CC1 gene expression was compared for cases in which doxorubicintreatment was conducted for parental U-2 OS cells, MDR variants of U-2OS cells (U-2 OS/DX580), and U-2 OS cells introduced with MDR1 gene(U-2/DOXO35), whereby it was found that in the parental U2 OS cells andcontrol cells introduced with a gene (U-2/Neo8) doxorubicin loweredexpression of the RB1CC1 gene and induced cell death. In contrast, inthe MDR variants of U-2 OS cells, doxorubicin treatment did not exhibitan inhibitory effect on the expression level of RB1CC1 gene, celllifetime, or cell proliferation, and in cells with the MDR1 gene theRB1CC1 gene expression was increased. In these cells, RB1CC1 geneexpression and RB1 gene expression correlated, and expression of bothgenes sustained the proliferation of these cells.

To examine the relation between expression of RB1 gene and the RB1CC1gene of the present invention, expression of both genes in 5 kinds ofMDR-variants of U-2 OS human osteosarcoma cells and 24 kinds of humantumor cells (10 kinds of osteosarcoma, 4 kinds of lung cancer, 7 kindsof breast cancer, 3 kinds of blood cancer) was examined, whereby it wasfound that RB1CC1 gene expression strongly correlated with RB1 geneexpression in all of the cells. Expression of RB1CC1 gene and RB1 genealso showed a similar correlation in Northern blot analysis ofnonneoplastic tissue.

Further, exogenous expression of the RB1CC1 gene of the presentinvention increased RB1 gene expression in K562 cells and Jurkat cells.Expression of MDR1 gene could not be detected in these cells. Inductionof RB1CC1 gene also stimulated transcriptional activity of RB1 genepromoter. Introduction of the RB1CC1 gene raised expression of RB1 genethrough the stimulated activity of the RB1 gene promoter.

Considering the amino acid sequence of the novel protein RB1CC1, thenuclear locality thereof, and the expression pattern thereof, there is apossibility that the RB1CC1 gene of the present invention is atranscription factor that enhances RB1 gene expression directly orindirectly through a molecular intermediate. While analysis of promotersequences of RB1 genes derived from human and mouse indicates thepossibility of the presence of a constitutive transcription factor suchas Sp1 or ATF, a transcription factor that directly regulates RB1 geneexpression is not known. In about 80% of human cancers, molecules thatare present in the RB1 gene pathway are associated with the mechanism ofcarcinogenesis, and dysregulation of the RB1 gene plays an importantrole in the cancer of many people.

As shown in Table 1, human and mouse RB1CC1 genes of the presentinvention both contain 24 exons and 23 introns, and length 4 kb or moreand 57 kb or more in human and mouse, respectively. A translationinitiation position is present at the site of exon 3. The structure ofthe gene in mouse was clarified using primers set forth in SEQ ID Nos:84 to 132 of the sequence listing. When we investigated the localizationsites of the gene on a chromosome, we found that the gene is present at8q11.2 on the chromosome 8 in human and at 1A2-4 on the chromosome 1 inmouse. TABLE 1 Structure of RB1CC1 gene Exon Intron nucleic acid nucleicacid strand length (bp) strand length (kb) Human Sequence No. humanmouse No. human mouse receptor sequence in splicing donor sequence insplicing 1 358 298 1 9.1 11.2 GCGTTGCCGG gtaagtgtcg 2 115 110 2 1.3 1.8tcttttccag TTTTCTCAGT GTGCCTGACG gtaagtcaca 3 122 115 3 1.4 3.5tttcttctag TAACTGTATC CAGTGCAAAC gtaagttgta 4 127 127 4 0.2 0.1ttttttgaag TGTGGCAGAC TGCTGGGACG gtaggtattc 5 171 171 5 7.0 3.8aaaaatatas GATACAAATC GCTTGCATTG gtaagatata 8 203 203 8 2.1 1.3ttcaatatag GAAATGTATG AACTTACTCA gtatgtttgc 7 430 427 7 5.7 3.8gtattttaag TTTAGGAACT TATGAGCAGG gtaagtaacg 8 171 171 8 6.3 0.5tgtcatttag CTTGATCCAA GCTTGCTCAG gtacctattt 9 185 185 9 0.3 0.2tttctcaaag GGATTTTTAG TCAGACTGAA gtaagtgatt 10 187 187 10 0.1 0.1tattctctag GTGGTGTTGC CTACAGGGAG gtatgcaagt 11 82 82 11 0.3 0.1cctcttctag TGGGCTGGTG AAATTATTTA gtaagtgttc 12 62 62 12 1.6 1.8ctttatacag GGAAGTCTTT TTCCTTTTGT gtatgtattt 13 104 104 13 0.8 0.3tttggtacag ACTCAAAAGC CATTCCTCAG gtaaatgtca 14 127 127 14 0.1 0.1tctgtttcag GGTTCCCTTA TGAACAAAAG gcaaattcaa 15 1801 1892 15 10.1 10.0tgttttccag GCATCTGTGA TAGCAAAAAG gtaagaatta 16 166 166 16 2.9 1.6aatttgtaag TCCTGCCATT GGAACAACAG gtctgtatct 17 109 109 17 0.1 0.1cttsttccag ACCATTTTA CGGGATAAAG gtttgtactg 18 241 241 18 6.3 1.1tgtccttcag ATTTGATAGA TGTCTGTACA gtaagtatgg 19 55 49 19 1.0 1.0tcacttttag AGAAAATATT GTTAGAACGA gtaagtaaat 20 48 48 20 4.4 3.0ccacctgcag ACATTGCAAT TCAAAGACTG gtaagatttt 21 59 59 21 2.3 2.1ttttttttag ATGTCTCAGA CTATTAGAGA gtaagtattt 22 137 137 22 3.5 2.0ctttattcag TTTTCAGGTG GGTGAGGGTG gtaagtgtca 23 71 71 23 0.8 1.6atttcattag CTTCAGGTGC AGCCAAAAAG gtaaaaacga 24 1401 1379 tccctcttagGCACAAAACAExon sequences are shown in upper case letters, and intron sequences areshown in lower case.

In order to detect mutations of RB1CC1 gene of the present invention,the RB1CC1 gene was analyzed using cDNA prepared from 35 cases ofprimary breast cancer, whereby 9 kinds of mutation were verified in 7 ofcancers. There were lacks at exons 3 to 24 in all of 9 kinds ofmutation, and the fragmented novel protein RB1CC1 had lost its consensusnuclear localization signal sequence site, leucine zipper motif sequencesite and coiled-coil structure, and did not have functions of thefundamental novel protein RB1CC1.

Two of primary breast cancers (MMK 3 and 6) showed compound heterozygouslacks in both alleles, and it is predicted that a clearly fragmentednovel protein RB1CC1 can be obtained from RB1CC1 gene with a lack. InMMK 6, there were lacks at exons 3 to 24 (nucleotides 534-5322) andexons 9 to 23 (nucleotides 1757-5187), with the respective frameshiftingat codons 4 and 411. In MMK 3, there were lacks at exons 3 to 24(nucleotides 535-5324) and exons 5 to 11 (nucleotides 849-2109), withtermination occurring at codon 4 in the former, and a frame shift causedat codon 109 in the latter to result in obtainment of a protein fragmentcomprising 122 amino acids. Although irregular products corresponding torespective lack mutations were detected in PCR of genome DNA of cancersamples, mutations were not observed in DNA of embryonic cells,revealing that these mutations occur in somatic cells. The novel proteinRB1CC1 was not detected in these cancers, and RB1 protein was absent inMMK 6 and was significantly less abundant than normal in MMK 3. Therewas no loss of heterozygosity at the RB1 loci on the chromosome ineither case. In the cancer samples (MMK 12 and 29) without mutation ofthe RB1CC1 gene, both the novel protein RB1CC1 and RB1 protein werepresent. This suggests that inactivated mutation of the RB1CC1 genecauses RB1 gene expression to be insufficient and promotes dysregulationof the RB1 gene pathway, to cause canceration.

In other five breast cancers, (MMK 1, 15, 31, 38 and 40) also, lackswere detected in RB1CC1 gene that generated a protein fragment withoutfunction. These mutations were all heterozygotes, with loss ofheterozygosity also present at the RB1CC1 loci, and since there was noexpression of RB1CC1 gene in each of the cases, it was suggested thatloss of function had occurred in both alleles. Expression of RB1 proteinin these cancers was clearly reduced in comparison to cases (MMK 12 and29) without mutation of RB1CC1 gene and RB1 gene. Loss of heterozygosityat the RB1 loci was not observed in these 5 cancers (MMK 1, 15, 31, 38,and 40).

Homozygous inactivation of the RB1CC1 gene of the present invention isassociated with genesis of breast cancer. Lack mutations of the RB1CC1gene that generated fragments of the novel protein RB1CC1 that clearlyhad no function were observed in approximately 20% of primary breastcancers examined. Two of these cancers showed plural heterozygous lackswithin the RB1CC1 gene, and the remainder showed loss of heterozygosityof the RB1CC1 gene. Although the novel protein RB1CC1 could not bedetected in any of seven cancers, protein was expressed in cancerswithout mutation of the RB1CC1 gene. Irrespective of the fact that therewas no loss of heterozygosity at the RB1 loci, in all seven cancers theRB1 protein was either absent or significantly decreased.

The novel protein RB1CC1 performs regulation to increase expression ofthe RB1 gene, and the RB1CC1 gene functions as a tumor suppressor inbreast cancer. Further, abnormality or inactivation of the RB1CC1 geneleads to a decline in expression of RB1 gene, causing genesis andprogression of cancer.

As described in the above-mentioned, since expression of the RB1CC1 geneand protein correlate with expression of RB1 gene, a more useful methodof diagnosing cancer cells or cancer can be provided by performing teststhat combine testing for the RB1CC1 gene and protein of the presentinvention with testing for expression of the RB1 gene or expression ofthe protein.

Further, by also combining tests for multidrug resistance gene (MDR1) orthe protein thereof, the effect of a pharmaceutical against a cancer orcancer cells can be examined, enabling the provision of an examinationmethod or a diagnostic method that is useful for selecting an anticanceragent and predicting the effects thereof.

(Polypeptide or Protein)

The novel protein of the present invention is a polypeptide or proteincomprising an amino acid sequence represented by SEQ ID No: 1 or 2 inthe sequence listing. The polypeptide or protein of the presentinvention may also be selected from polypeptides having a partialsequence of the polypeptide represented by SEQ ID No: 1 or 2 in thesequence listing. The selected polypeptide preferably has homology ofabout 70% or more, more preferably about 80% or more, andfurtherpreferablyhas homologyexceeding about 90% with thepolypeptiderepresentedby SEQID No: 1 or 2 in thesequence listing. Selection ofpolypeptides having the homology can be conducted, for example, bytaking expression of RB1 gene or RB1 protein as an indicator.

Techniques for determining homology of an amino acid sequence arepublicly known in the art and, for example, a method that directlydetermines the amino acid sequence or a method that first determines aputative base sequence of a nucleic acid and then predicts the aminoacid sequence encoded by the base sequence may be used.

For the polypeptide of the present invention, an amino acid sequenceselected from polypeptides having a partial sequence of a polypeptide orprotein comprising an amino acid sequence set forth in SEQ ID No: 1 or 2in the sequence listing can be utilized as a reagent, reference materialor immunogen. The subject of the present invention is a polypeptidecomprising, as a minimum unit thereof, the amino acid sequence composedof at least 5 amino acids, preferably at least 8 to 10 amino acids ormore, and more preferably at least 11 to 15 or more amino acids whichcan be screened immunologically.

Further, by employing expression of RB1 gene or RB1 protein as theindicator, there can also be provided a polypeptide comprising an aminoacid sequence having a mutation or induced mutation such as a deletion,substitution, addition or the like of one to several amino acidsrelative to the amino acid sequence of a polypeptide specified asdescribed above. Methods for carrying out a deletion, substitution,addition or insertion are publicly known, and, for example, thetechnique of Ulmer (Science, 219: 666, 1983) can be utilized. Theseavailable peptides can also be modified to a degree that is notaccompanied by a noticeable change in function, such as modification ofconstitutive amino groups or carboxyl groups or the like.

Polypeptides of the present invention can be used as they are in apharmaceutical composition for regulating a function of the novelprotein RB1CC1. Further, the polypeptide or protein of the presentinvention can be used in screening to obtain a compound that canregulate a function of the novel protein RB1CC1, for example, aninhibitor, antagonist, activator or the like, or an antibody against thenovel protein RB1CC1. In addition, a polypeptide or protein of thepresent invention can also be used as a reagent or reference standard.

(Nucleic Acid)

The term “nucleic acid and a complementary strand thereof” of thepresent invention refers to a nucleic acid set forth in SEQ ID No: 3 or4 in the sequence listing that codes for an amino acid sequence setforth in SEQ ID No: 1 or 2 in the sequence listing and the complementarystrand for the nucleic acid, a nucleic acid hybridizing under stringentconditions with these nucleic acids, and a nucleic acid having asequence of at least 15 consecutive base sequence derived from thesenucleic acids in which a peptide encoded thereby is capable of bindingwith an antibody against the novel protein RB1CC1. When DNA is taken asa typical example of the nucleic acid, the term “DNA hybridizing understringent conditions to DNA” refers to DNA that can be obtained by apublicly known method, for example, a method described in MolecularCloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press,1989). Here the term “hybridizing under stringent conditions” refers to,for example, conditions under which a positive hybridization signal isstill observed even after heating at 42° C. in a solution of 6×SSC, 0.5%SDS and 50% formamide, and washing at 68° C. in a solution of 0.1×SSCand 0.5% SDS.

The term “nucleic acid of the present invention” refers to a homologousstrand and complementary strand selected from information of the nucleicacid set forth in SEQ ID No: 3 or 4 in the sequence listing that encodesan amino acid sequence described in SEQ ID No: 1 or 2 in the sequencelisting, and also refers to a nucleic acid sequence comprising asequence of at least about 15 to 20 nucleotides that correspond to aregion of the specified nucleotide sequence, as well as thecomplementary strand thereof. Determination of this useful nucleic acidsequence can be conducted by simply confirming the expressed proteinutilizing a publicly known protein expression system, for example, acell-free protein expression system, and then screening by employingbinding thereof with the antibody against bioactive novel protein RB1CC1as the indicator. As the cell-free protein expression system, forexample, a ribosome system derived from germ or rabbit reticulocyte orthe like can be utilized (Nature, 179, 160-161, 1957).

Each of these nucleic acids provide genetic information that is usefulfor producing the novel protein RB1CC1 of the present invention and thepolypeptide or protein of the present invention, and they can be used asprimers or probes for detecting mRNA or a nucleic acid such as a geneencoding these, or as antisense oligomers to regulate gene expression.Further, a nucleic acid of the present invention can also be utilized asa reagent or reference standard relating to the nucleic acid.

(Transformant)

In addition to the cell-free protein expression system described above,by employing genetic recombination techniques using a publicly knownhost such as Escherichia coli, yeast, Bacillus subtilis, an insect cellor animal cell, it is possible to provide the novel protein RB1CC1comprising the present invention and the polypeptide comprising aproduct derived therefrom.

Transformation can be conducted by applying publicly known means, forexample, by transforming the host utilizing a plasmid, chromosome, virusor the like as a replicon. As a more preferable system, a method thatconducts integration into the chromosome may be mentioned whenconsidering genetic stability. However, as a simple and convenientmethod, an autonomous replication system using an extra nuclear gene canbe utilized. A vector can be selected according to the kind of host, andgene sequences that are objects of expression and gene sequencescarrying information relating to replication and regulation can beemployed as constituent elements. Constituent elements can be selectedaccording to whether the host is a prokaryotic cell or eukaryotic cell,and a promoter, ribosome binding site, terminator, signal sequence,enhancer and the like can be combined according to a publicly knownmethod and used.

The transformant can be used to produce the polypeptide of the presentinvention by culturing the transformant after selecting optimalconditions from publicly known culture conditions for the respectivehosts. While culturing may be conducted by employing as an indicator thephysiological activity of the novel protein RB1CC1 to be expressed andproduced and a polypeptide comprising the product derived therefrom, inparticular, RB1 gene inducing activity or DNA-binding transcriptionfactor activity, it is generally conducted by subculture or batchculture employing the quantity of transformant in the medium as anindicator.

(Recovery of the Novel Protein RB1CC1 and Product Derived Therefrom)

Recovery from the culture medium of the novel protein RB1CC1 and thepolypeptide comprising the product derived therefrom can be conducted bycarrying out purification and recovery that combines techniques such asa molecular sieving, an ion column chromatography, an affinitychromatography employing binding with the antibody against the novelprotein RB1CC1 as the indicator, or by a fractionation technique usingalcohol or ammonium sulfate or the like that is based on difference insolubility.

(Antibody)

An antibody can be prepared by screening for an antigenic determinant ofthe novel protein RB1CC1 of the present invention and the polypeptidecomprising the product derived therefrom. The antigenic determinant iscomposed of at least five amino acids, and more preferably at least 8 to10 amino acids. The amino acid sequence need not necessarily behomologous with SEQ ID No: 1 or 2 in the sequence listing, and it issufficient that the sequence is a site that is exposed to outside of thetertiary structure of the protein. If the exposed site is adiscontinuous site, it is also effective that the amino acid sequencethat is continuous with respect to the exposed site. The antibody is notparticularly limited as long as it immunologically recognizes the novelprotein RB1CC1 and the polypeptide comprising the product derivedtherefrom. The presence or absence of the recognition can be determinedby a publicly known antigen-antibody binding reaction.

Production of the antibody can be conducted by inducing immunity such ashumoral response and/or cellular response in an animal using the novelprotein RB1CC1 of the present invention and the polypeptide comprisingthe product derived therefrom by itself or in a state in which it isbonded with a carrier, in the presence or absence of an adjuvant. Thecarrier is not particularly limited as long as the carrier itself doesnot produce a deleterious effect on a host, and examples thereof includecellulose, polymerized amino acid, and albumin. As an animal to beimmunized,mouse, rat, rabbit, goat, horse or the like is preferable. Apolyclonal antibody can be obtained by a publicly known method forrecovering antibody from serum.

Production of a monoclonal antibody can be carried out by recoveringantibody-producing cells from the animal that has undergone theaforementioned immunization and introducing transformation means topublicly known constantly proliferating cells.

The polyclonal or monoclonal antibody can be bonded directly with thenovel protein RB1CC1 of the present invention to enable control of theactivity thereof, and control of expression of the novel protein RB1CC1and RB1 gene or protein can be easily performed. Therefore, the antibodyis useful for treating or preventing a disease with which the RB1 geneproduct and the novel protein RB1CC1 are associated.

(Screening)

According to the novel protein RB1CC1 and the polypeptide comprising theproduct derived therefrom that were prepared as described above, thenucleic acid encoding these and a complementary strand thereof, the celltransformed based on information of these amino acid sequences and basesequences, and the antibody that immunologically recognizes the novelprotein RB1CC1 and the polypeptide comprising the product derivedtherefrom, by use of a single means or by combining a plurality ofmeans, there can be provided means effective in screening for bindingwith the novel protein RB1CC1 and the polypeptide comprising the productderived therefrom, a function of the novel protein RB1CC1, or aninhibitor or activator of expression of the novel protein RB1CC1. Morespecifically, there can be provided a method of screening for compoundsthat inhibit or enhance expression of the polypeptide or protein and theRB1 gene or protein of the present invention by using at least onemember of the group consisting of the polypeptide of the presentinvention and the antibody of the present invention. There can beprovided a method of screening for compounds that interact with thenucleic acid of the present invention to inhibit or enhance expressionof the nucleic acid by using at least one member of the group consistingof the nucleic acid of the present invention, vector of the presentinvention, transformant of the present invention, and antibody of thepresent invention. There can be provided a method of screening forcompounds that inhibit or enhance a function of the polypeptide orprotein of the present invention to regulate expression of the RB1 geneor protein by using at least one member of the group consisting of thepolypeptide or protein of the present invention and the antibody of thepresent invention. For example, screening for the antagonist obtained bydrug design based on the tertiary structure of the polypeptide,screening for an expression regulator at the genetic level that utilizesa protein expression system, screening for an antibody recognizingsubstance utilizing the antibody and the like can be utilized in apublicly known pharmaceutical screening system.

(Compound, Pharmaceutical Composition)

Compounds obtained by the above-described screening methods can beutilized as candidate compounds for the inhibitor, antagonist, activatoror the like that regulates a function of the novel protein RB1CC1 andthe polypeptide comprising the product derived therefrom to controlexpression of RB1 gene or protein. Compounds can also be utilized ascandidate compounds for an inhibitor, antagonist, activator or the likefor expression of the novel protein RB1CC1 and the polypeptidecomprising the product derived therefrom at the genetic level. Examplesof aforementioned candidate compounds for an inhibitor, antagonist,activator or the like include a protein, a polypeptide, a polypeptidewithout antigenicity, and a low molecular weight compound, and a lowmolecular weight compound is preferred.

Candidate compounds that were screened in the above manner can beselected in consideration of a balance between biological usefulness andtoxicity to be prepared as pharmaceutical compositions to be used fortreatment of osteosarcoma, leukemia or a tumor originating from themammary gland, prostate gland, lung, or colon or the like. Further, thenovel protein RB1CC1 comprising the present invention and thepolypeptide comprising the product derived therefrom, nucleic acidsencoding these and complementary strands thereof, vectors containingthese base sequences, and antibodies that immunologically recognize thenovel protein RB1CC1 and the polypeptide comprising the product derivedtherefrom can be used as pharmaceutical means, by themselves, that havean inhibitory, antagonizing or activating function with respect tointeraction between the novel protein RB1CC1 and RB1 gene product andare used in treatment of breast cancer, prostrate cancer and the like .Here, the term “breast cancer, prostrate cancer and the like” includes abenign tumor and a malignant tumor, and in this connection, at the timeof formulation, publicly known formulation means may be introduced inaccordance with the substance for formulation, such as the polypeptide,protein, nucleic acid or antibody.

The novel protein RB1CC1 of the present invention and the polypeptidecomprising the product derived therefrom, nucleic acids encoding theseand complementary strands thereof, vectors containing these basesequences, and antibodies that immunologically recognize the novelprotein RB1CC1 and the polypeptide comprising the product derivedtherefrom can be used as means for testing or diagnosing a disease withwhich expression of the polypeptide of the present invention or theactivity thereof is related, such as a disease relating to expression ofthe novel protein RB1CC1 of the present invention or interaction withRB1 gene or the product thereof. In particular, they are useful as meansfor examination and diagnosis such as a diagnostic marker and/or reagentor the like for breast cancer, prostrate cancer and the like. Diagnosiscan be conducted by utilizing interaction or reactivity with the nucleicacid sequence encoding the novel protein RB1CC1 to determine theabundance of a nucleic acid sequence of interest, and/or determine thebiodistribution for the novel protein RB1CC1, and/or determine theabundance of the novel protein RB1CC1 in a test sample. Morespecifically, testing can be conducted utilizing the novel proteinRB1CC1 as the diagnostic marker. As a method of determination, apublicly known antigen-antibody reaction system, enzyme reaction system,PCR reaction system or the like may be used. Further, a reagent kit orthe like used in a method of examination and diagnosis is also included.

EXAMPLES

The present invention is described in further detail hereunder on thebasis of examples, however, the present invention is not limited by thefollowing examples.

Example 1 cDNA of Human RB1CC1

In order to identify genes involved in MDR, we found a gene thatexpresses differentially in U-2 OS osteosarcoma cells and MDR-variantinduced cells, to thereby identify a novel human gene. The gene wascloned using the set of primers (CC1-S1 and CC1-AS1) set forth in SEQ IDNos: 5 and 26 and the set of primers (CC1-S2 and CC1-AS2) set forth inSEQ ID Nos: 6 and 25 in the sequence listing, and the nucleic acidsequence thereof was then determined using the primers set forth in SEQID Nos: 7 to 24. Further, the cDNA sequences at the 5′- and 3′-ends wereidentified using a commercially available rapid amplification kit forcDNA end sequences (RACE kit, manufactured by Roche) and the primers setforth in SEQ ID Nos: 27 to 37. The DNA and the amino acid sequenceencoded thereby were analyzed using DNAs is Version 3.2 SequenceAnalyzer (manufactured by Hitachi Software Engineering Co.) and PSORT II(http://www.yk.rim.or.jp/˜aisoai/molbio-j.html). Results showed that thecDNA had a length of 6.6 kb including an open reading frame (ORF) of4782 nucleotides, encoding a protein comprising 1594 amino acids with amolecular weight of 180 kDa.

Example 2 cDNA of Mouse Rb1cc1

The mRNA of mouse muscle was employed as a template for amplification byRT-PCR, and cloning was then conducted using the set of primers (MCC1-S1and MCC1-AS1) set forth in SEQ ID Nos: 53 and 73 and the set of primers(MCC1-S2 and MCC1-AS2) set forth in SEQ ID Nos: 54 and 72 in thesequence listing. The nucleic acid sequence was determined using primersset forth in SEQ ID Nos: 55 to 71 in the sequence listing. The cDNA ofnovel mouse protein Rb1cc1 was then identified using a similar method toExample 1, with the exception that rapid amplification of the cDNA wasconducted using the primers (MCC-ASR1, MCC-ASR2, MCC-ASR3 andINTRON1ASR) set forth in SEQ ID Nos: 74 to 77 in the sequence listing asprimers for the 5′-end RACE, and the primers (MCC-SR1, MCC-SR2, MCC3-S3,MCC3-S4, MCC3-AS2 and MCC3-AS3) set forth in SEQ ID Nos: 78 to 83 asprimers for the 3′-end RACE. The cDNA encoding novel mouse proteinRb1cc1 has a strand length of 6518 bp including an open reading frame(ORF) of 4764 bp encoding 1588 amino acids. The gene of novel mouseprotein Rb1cc1 had homology of 86% at the nucleic acid level and 89% atthe protein level with the gene of novel human protein RB1CC1 (see SEQID Nos: 1 to 4).

Example 3 Analysis of MDR1 Gene and RB1CC1 Gene of the Present Invention

Expression levels of RB1CC1 gene and MDR1 gene in parental U2 OS cellsand several kinds of MDR-variant cells were analyzed by Northernblotting. A probe hybridizing between nucleotide numbers 4190 and 4654of the RB1CC1 gene sequence was used as a probe for analysis of RB1CC1gene, and a probe hybridizing between nucleotide numbers 834 and 1119 ofMDR1 gene was used for MDR1 gene. Probes were used after labeling withα-32P-dCTP in which phosphorus at an alpha position ofdeoxycytidine-3-phosphate was substituted with a radioactive isotope.Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as theindicator of mRNA expression. The results showed that the expressionlevels of both genes correlated inversely (FIG. 1).

Example 4 Preparation of Antibody and Western Blot Analysis

Three kinds of synthetic polypeptide were prepared which respectivelycomprised amino acids 642 to 658 (RB1CC-642), 744 to 757 (RB1CC-744) and1104 to 1118 (RB1CC-1104) of the amino acid sequence of the novelprotein RB1CC1 of the present invention. Rabbits were immunized by aconventional method with substances in which a cysteine residue had beenintroduced at the amino terminus of each polypeptide, and antibody wasthen obtained. After subjecting nuclear components and cytoplasmiccomponents of U-2 OS cells to SDS-PAGE, respectively, analysis wascarried out by Western blotting using the antibody prepared above.Results showed that RB1CC1 protein of a molecular weight of 180 kDa waspresent in the nucleus (FIG. 2).

After subjecting nuclear components and cytoplasmic components ofNIH3T3-3 cells of mouse to electrophoresis in a similar manner, Westernblot analysis was conducted using the RB1CC-642 antibody. Detection ofstathmin was simultaneously conducted using anti-stathmin rabbitantibody. Results showed that the Rb1cc1 protein is localized in thenucleus, while stathmin is present in cytoplasm. When same cells weresubjected to immunocytochemical staining using each antibody and thencompared, it was found that while the nucleus was stained with theRB1CC-642 antibody, the cytoplasm was stained with the anti-stathminrabbit antibody (FIG. 3).

Above results showed that the novel protein RB1CC1 of the presentinvention is present in the nucleus of mammalian cells.

Example 5 Effect of Anticancer Agent on Expression of RB1CC1 Gene of thePresent Invention

The influence of an anticancer agent was assessed for 4 kinds of cellsthat were treated with doxorubicin, including parent cells (U-2 OS), MDRvariants of U-2 OS cells (U-2 OS/DX580) and U-2 OS cells introduced withthe MDR1 gene (U-2/DOXO 35). The effect on cell proliferation in thepresence of 450 ng/mL of the anticancer agent doxorubicin was examined.As shown in FIG. 4, results indicated that while cell proliferation wassuppressed by the anticancer agent in parental U2 OS cells and controlcells introduced with a gene (U-2/Neo8), the anticancer agent had noeffect on MDR variants of U-2 OS cells (U-2 OS/DX580) and U-2 OS cellsintroduced with MDR1 gene (U-2/DOXO 35) and cell proliferation continuedfor 120 hours or more (FIG. 4).

mRNA expression levels of cells that were obtained over time in theabove-mentioned experiment were analyzed. Analysis was conducted for thenovel gene RB1CC1 gene of the present invention, the RB1 gene and theMDR1 gene, respectively, in the same manner as Example 3 with theexception that expression levels of the RB1 gene were detected using aprobe hybridizing to the site at nucleotides 336 to 675 of thenucleotide sequence of human RB1 mRNA. Results are shown in FIG. 5. Forparental U2 OS cells and control cells introduced with a gene (U-2/Neo8)for which the effect of the anticancer agent was observed, expression ofthe RB1CC1 gene decreased over time. In contrast, in MDR variants of U-2OS cells (U-2 OS/DX580) and U-2 OS cells introduced with MDR1 gene(U-2/DOXO 35), expression level of RB1CC1 gene was not inhibited bytreatment with doxorubicin, and expression of RB1CC1 gene increased. Inthese cells, RB1CC1 gene expression and RB1 gene expression correlated(FIG. 5).

Example 6 Expression of RB1 Gene and RB1CC1 Gene of the PresentInvention

The expression of RB1CC1 gene and RB1 gene in various cancer cells wasassessed by semi-quantitative RT-PCR. Cell lines used were SARG, IOR/OS9, 10, 14, 15, 18, MOS (these were obtained from surgical samples ofadvanced human osteosarcoma), Saos-2, HOS, MCF-7, T-47D, BT-20, SK-BR3,ZR75-1, MDA-MB-231, Daudi, Jurkat and K562 (these were purchased fromthe American Type Culture Collection), NZK-K1 (this was established frombreast cancer tissue of a 46-year old female), LK2, QG56, EBC1 and SBC2(these were provided by Doctor Tatsuhiko Narita of Aichi Cancer Center).2 μg of RNA was extracted from each cell line, and subjected to 22 to 30cycles of RT-PCR for amplification. Publicly known primers weresynthesized and used as primers for the RB1 gene (Sauerbrey et al.,1996). The combination of primers set forth in SEQ ID Nos: 19 and 20 inthe sequence listing (CC1-S and CC1-AS) were used as primers foramplification of RB1CC1. β₂-microglobulin was used as a control. In allof these cells, expression of RB1CC1 gene correlated closely with thatof RB1 gene. FIG. 6 shows results for one case of normal leukocyte andsix cancer cells: T-47D, MCF7, NZK-K1, Daudi, K562 and Jurkat (FIG. 6).

Example 7 Expression of RB1CC1 Gene and RB1 Gene of the PresentInvention in Organs

Northern blot analysis was conducted for RB1CC1 gene and RB1 geneexpressing in nonneoplastic tissue of human brain, heart, skeletalmuscle, colon, thymus, spleen, kidney, liver, small intestine, placenta,lung and leukocyte, respectively, using commercially available MTN Blots(manufactured by Clontech). Results are shown in FIG. 7. Both genes wereexpressed strongly in heart and skeletal muscle, while expression wasweak in colon, small intestine, lung and leukocyte. However, expressionof RB1CC1 gene and RB1 gene correlated. Northern blot analysis was alsoconducted for Rb1cc1 gene expressing in respective tissues of heart,brain, spleen, lung, liver, skeletal muscle, kidney and testis of mouse.Results are shown in FIG. 8. Transcription products of 6.2 kb and 6.8 kbwere expressed strongly in heart, while expression was observed to acertain extent in kidney, liver and skeletal muscle. The principalexpression in testis was 6.2 kb, while expression was weak in lung andspleen (FIG. 7, FIG. 8).

Example 8 Expression of RB1 Gene Induced by Introduction of RB1CC1 Geneof the Present Invention

Jurkat and K562 cells that had weak expression levels for both RB1CC1gene and RB1 gene among cells shown in Example 6 were subjected toexogenous introduction of RB1CC1 gene to examine changes in theexpression of RB1 gene. A 4.9-kb gene that included the complete codingregion of the RB1CC1 molecule was incorporated into pCR3.1-Uni vector(manufactured by Invitrogen), which was then cloned to prepare an RB1CC1expression vector (pCR-RB1CC). The thus-prepared expression vector wasincorporated into K562 and Jurkat cells to prepare RB1CC1 transformedcells. A control was prepared by incorporating lac Z gene intopCR3.1-Uni vector. Respective expression levels of RB1CC1 gene and RB1gene in parent cells and transformed cells (cells introduced with RB1CC1gene) were examined in a similar manner to Example 6. FIG. 9 shows theresults. Although expression of both RB1CC1 gene and RB1 gene was weakin untransformed cells and cells into which the lac Z gene wasincorporated, it was found that in cells incorporated with RB1CC1 gene,the RB1CC1 gene expression was strong as expected and the RB1 gene wasalso strongly expressed, showing that expression of the RB1 gene wasalso induced by introduction (exogenous expression) of the RB1CC1 gene(FIG. 9).

Example 9 RB1 Gene Promoter Transcriptional Activity of RB1CC1 Gene ofthe Present Invention

We examined whether introduction of the RB1CC1 gene enhanced thetranscriptional activity of the promoter region of RB1 gene. A gene ofRB1 promoter region of approximately 2 kb was amplified with the pair ofprimers 5′-GAA GAT CTT TGA AAT TCC TCC TGC ACC A-3′ (Bg1.RbPro-S) and5′-CCC AAG CTT AGC CAG CGA GCT GTG GAG-3′ (Hind.RbPro-AS), andincorporated into PicaGene Basic vector 2 (manufactured by Toyo InkMfg.Co., Ltd.). Then, RB1 promoter which controls expression of fireflyluciferase was used to prepare pGV-RbPro vector. The prepared pGV-RbProvector was then retranscribed with pRL-SV40 encoding the sea pansyluciferase gene, as an internal control, and incorporated into K562 cellusing LIPOFECTAMINE PLUS reagent (manufactured by GIBCO-BRL). Results ofanalysis conducted after 48 hours using a double luciferase assay system(Toyo Ink Mfg. Co., Ltd.) showed that K562 cell introduced with RB1CC1gene exhibited strong luciferase activity compared to K562 cellincorporated with lac Z as a control, showing that introduction of theRB1CC1 gene enhanced the transcriptional activity of RB1 gene promoter(FIG. 10).

Example 10 Loss of Heterozygosity at Locus (D8S567) of RB1CC1 Gene inPrimary Breast Cancer

DNA samples of cancer tissue and genome DNA from same patients wereamplified by PCR and the amplification products were analyzed using 8%urea-denatured polyacrylamide gel electrophoresis. Results obtained bysilver staining after electrophoresis are shown in FIG. 11. While twobands were observed for the genome DNA of each patient to indicateretention of heterozygosity, only one band was detected in five cases ofDNA of cancer tissue, indicating loss of heterozygosity (FIG. 11).

Example 11 Analysis of Mutation of RB1CC1 Gene of the Present Inventionin Breast Cancer

Mutations of RB1CC1 gene were identified by analyzing the geneticsequence of cDNA samples that were amplified using ELONGASE System(manufactured by GIBCO-BRL) with the pair of primers (CC1-S2 andCC1-AS2) set forth in SEQ ID Nos: 6 and 25 that were used in Example 1,using ABI PRISM 310 genetic analyzer and the primers set forth in SEQ IDNos: 7 to 24 in the sequence listing. As a result, 7 cases of mutationwere verified among 35 cases of breast cancer, and 9 kinds of variantswere verified. This result was reconfirmed using primers set forth inSEQ ID Nos: 38 to 52. Results are shown in Table 2. TABLE 2 Mutations ofRB1CC1 gene in primary breast cancer sample nucleotide locationpredicted genome State of RB1CC1 gene State of RB1 name mutation (exon)influence DNA allele protein LOH protein MMK3 c.11_4800del 3-24 Y4fsX4wild type plural heterozygous deletions (−) (−) ↓ ↓ c.325_1585del 5-11P109fsX122 wild type plural heterozygous deletions (−) (−) MMK6c.10_4798del 3-24 Y41sX48 wild type plural heterozygous deletions (−)(−) (−) c.1233_4633del 9-23 D411fsX431 wild type plural heterozygousdeletions (−) (−) MMK1 c.957_4785del 7-24 N319fsX368 wild type pluralheterozygous deletions (−) (−) ↓ ↓ MMK15 c.1635_4719del 12-24 S545fsX557wild type plural heterozygous deletions (−) (−) (−) MML31 c.212_4188del5-24 171fsX111 wild type plural heterozygous deletions (−) (−) (−) MMK38c.241_4621del 5-22 O81fsX99 wild type plural heterozygous deletions (−)(−) ↓ ↓ MMK40 c.591_4678del 7-23 S197fsX212 wild type pluralheterozygous deletions (−) (−) ↓ ↓(−): absent,↓ ↓: significantly decreasedLOH: loss of heterozygosky

Example 12

FIG. 12 shows results of analysis of PCR products for MMK6 in whichmutation was observed in RB1CC1 gene and MMK29 in which mutation was notobserved among samples analyzed in Example 11, as well as the resultsof-genetic sequence analysis corresponding thereto. It was found that agene of 4.9 kb expressed in MMK29 that was without mutation, while the4.9-kb expression was not observed in MMK6 with mutation and expressionof gene fragments (1456 bp and 98 bp) was observed (FIG. 12).

Example 13 Analysis by Western Blotting

From the samples analyzed in Example 11, expression of the novel proteinRB1CC1 and the RB1 protein was verified by Western blotting in 3 cancers(MMK6, MMK40, MMK38) in which mutation was observed in RB1CC1 gene and 2cancers (MMK12, MMK29) in which mutation was not observed. Aftersubjecting extracted protein to 5% SDS-polyacrylamide gelelectrophoresis, and then transferring to PVDF membrane, reaction wasconducted with the anti-human RB1CC1 antiserum (α-RB1CC-642) prepared inExample 4. The RB1 protein was reacted with RB1 monoclonal antibody(G3-245, manufactured by PharMingen Inc.). After reaction, detection wascarried out using ECL reagent (manufactured by Amersham Biosciences) Theresults are shown in FIG. 13. While novel protein RB1CC1 having amolecular weight of 180 kDa and RB1 protein of a molecular weight of 110to 116 kDa both expressed in MMK12 and MMK29 without mutation, incontrast, expression of either protein was not observed in any of 3cancers with a mutation (FIG. 13).

Example 14 Immunohistological Staining

Immunohistological staining was conducted for 2 cancers (MMK3, MMK6) inwhich mutation in RB1CC1 gene was observed and 1 cancer (MMK 12) inwhich mutation was not observed among samples analyzed in Example 11.The antibody used for reaction was the same as that in Example 13, andthe antibody was reacted with tissue sections prepared from paraffinblocks obtained from each of cancer samples. As shown in FIG. 14, theexpression levels of novel protein RB1CC1 and RB1 protein correlated,and it was verified that expression levels were clearly lower in 2cancers (MMK3, MMK6) in which mutation in RB1CC1 gene was observedcompared to the cancer (MMK 12) in which mutation was not observed (FIG.14).

Example 15

54 samples of primary breast cancer tissue were assayed byimmunohistological staining in a similar manner to Example 14, and theRB1CC1 protein was not detected in 8 samples (corresponding to 15%).Then, RB1 protein expression was absent or significantly lowered in allof the samples.

For 46 cases expressing RB1CC1 protein, the RB1 protein wassimultaneously expressed in 45 cases. When the RB1 protein expressionwas compared with the RB1CC1 positive group and negative group by stainindication using immunohistological staining (indication showing as apercentage the ratio of the number of cells stained among 1000 or morecells), the RB1CC1 positive group and negative group were found to showa positive correlation with RB1CC1 expression, with 78.6±13.9% and13.6±12.1%, respectively (FIG. 15 a). Meanwhile, when immunohistologicalstaining for Ki-67 was conducted using mouse monoclonal antibody(NCL-Ki-67-MMI, manufactured by Novocastra Inc.), the stain indicationwas 20.3±12.8% for the RB1CC1 positive group and 65.0 ±12.2% for thenegative group, showing a clearly inverse correlation with RB1CC1expression (FIG. 15 b).

These results indicate that in cancers in which expression of RB1CC1protein is suppressed, the cell proliferation marker Ki-67 is expressedin large amounts, and proliferation of cancer cells flourishes. It wasthus found that assaying using a combination of RB1CC1 protein and Ki-67is useful for cancer diagnosis.

By testing for the novel gene (RB1CC1 gene) of the present invention andthe protein (RB1CC1) thereof, information that is useful for thediagnosis of cancer cell proliferation and cancer can be provided.

1. A protein or polypeptide which is present in nucleus of human oranimal cell and which has a transcription factor function and/or afunction that can induce expression of retinoblastoma gene (RB1 gene) ora gene product thereof.
 2. The human protein according to claim 1, whichis a polypeptide or protein selected from a group consisting of: (1) apolypeptide or protein represented by an amino acid sequence set forthin SEQ ID No: 1 in the sequence listing; (2) a polypeptide containing anamino acid sequence comprising at least five amino acids of the aminoacid sequence of the polypeptide or protein; (3) a polypeptide orprotein having homology of at least approximately 70% at the amino acidsequence level with the polypeptide or protein; and (4) a protein orpolypeptide having a mutation or induced mutation such as a deletion,substitution or addition of one to several amino acids relative to theamino acid sequence of the polypeptide or protein according to any oneof the preceding (1) to (3).
 3. The animal protein according to claim 1that is a protein derived from mouse, and which is a polypeptide orprotein selected from the group consisting of: (1) a polypeptide orprotein represented by an amino acid sequence set forth in SEQ ID No: 2in the sequence listing; (2) a polypeptide containing an amino acidsequence comprising at least five amino acids of the amino acid sequenceof the polypeptide or protein; (3) a polypeptide or protein havinghomology of at least approximately 70% at the amino acid sequence levelwith the polypeptide or protein; and (4) a protein or polypeptide havinga mutation or induced mutation such as a deletion, substitution oraddition of one to several amino acids relative to the amino acidsequence of the polypeptide or protein according to any one of thepreceding (1) to (3).
 4. A nucleic acid coding for the polypeptide orprotein according to claim 1, or a complementary strand thereof.
 5. Anucleic acid hybridizing under stringent conditions with the nucleicacid according to claim 3 or the complementary strand thereof.
 6. Anucleic acid represented by a base sequence comprising at least 15consecutive bases of the base sequence of a nucleic acid set forth inSEQ ID Nos: 3 to 4 in the sequence listing or a complementary strandthereof, wherein a polypeptide expressed by transcription of the nucleicacid is the polypeptide according to claim
 1. 7. A recombinant vectorcontaining the nucleic acid according to claim
 4. 8. A transformant thatwas transformed with the recombinant vector according to claim
 7. 9. Amethod for producing the polypeptide or protein according to claim 1,comprising a step of culturing the transformant with the recombinantvector containing nucleic acid coding for the polypeptide or protein.10. Nucleic acid primers set forth in SEQ ID Nos: 5 to 132 in thesequence listing, which hybridize under stringent conditions with thenucleic acid according to claim 4 or the complementary strand thereof.11. An antibody that immunologically recognizes the polypeptide orprotein according to claim
 1. 12. A method of screening for compoundsthat inhibit or enhance a function that can induce transcription factoractivity and/or expression of RB1 gene of the polypeptide or proteinaccording to claim 1, wherein the method utilizes the polypeptide, theprotein, or an antibody that immunologically recognizes the polypeptideor protein.
 13. A method of screening for compounds that interact withthe nucleic acid according to claim 4 to inhibit or enhance expressionof the nucleic acid, wherein the method utilizes the nucleic acid, arecombinant vector containing the nuclic acid a transformant that wastransformed with the recombinant vector, or nucleic acid primers setforth in SEQ ID NOS: 5 to 132 in the sequence listing which hybridizeunder stringent conditions with the nucleic acid.
 14. A compound thatwas screened by the screening method according to claim
 12. 15. Acompound that inhibits or enhances transcription factor activity and/ora function that can induce expression of RB1 gene of the polypeptide orprotein according to claim
 1. 16. A compound that interacts with thenucleic acid according to claim 4 to inhibit or enhance expression ofthe nucleic acid.
 17. A pharmaceutical composition for use in treatmentof multidrug resistance that is resistance to treatment with anticanceragents, wherein the pharmaceutical composition comprises the polypeptideor protein according to claim 1, a nucleic acid coding for thepolypeptide or protein or a complementary strand thereof, a recombinantvector containing the nucleic acid, a transformant that was transformedwith the recombinant vector, nucleic acid primers set forth in SEQ IDNOS: 5 to 132 in the sequence listing which hybridize under stringentconditions with the nucleic acid, an antibody that immunologicallyrecognizes the polypeptide or protein, or a compound that interacts withnucleic acid to inhibit or enhance expression of the nucleic acid.
 18. Amethod of testing and diagnosing a disease related with expression oractivity of the polypeptide or protein according to claim 1, wherein themethod comprises a step of conducting analysis employing (a) a nucleicacid encoding the polypeptide or protein and/or (b) the polypeptide orprotein, as a marker in a sample.
 19. The method of testing anddiagnosing according to claim 18, which is a method of testing cancercells or a method for diagnosing a cancer.
 20. The method according toclaim 18 which detects expression, increase, decrease, lack or the likeof all or a part of the polypeptide or protein, wherein the methodutilizes an antibody that immunologically recognizes the polypepetide.21. The method according to claim 18 which detects expression, mutation,lack or insertion or the like of all or a part of a gene encoding thepolypeptide or protein through a step of amplifying a gene encoding thepolypeptide or protein utilizing at least one of nucleic acid primersset forth in SEQ ID NOS: 5 to 132 in the sequence listing, whichhybridize under stringent conditions with the nucleic acid.
 22. Themethod according to claim 18, wherein the method combines assay ofexpression, increase, decrease, mutation, lack or insertion or the likeof all or a part of tumor-suppressor gene retinoblastoma gene (RB1 gene)or the gene product thereof (RB1 protein).
 23. The method according toclaim 18, wherein the method combines assay of expression, increase,decrease, mutation, lack or insertion or the like of all or a part ofmultidrug resistance gene (MDR1 gene) or the gene product thereof (MDR1protein: P-glycoprotein).
 24. The method according to claim 18, whereinthe method combines assay of expression, increase, or decrease or thelike of all or a part of the cell proliferation marker, Ki-67 protein.25. A method that tests drug sensitivity of a cancer cell using themethod according to claim
 23. 26. A kit and a reagent for assay ordiagnosis, for use in the method according to claim 18.